Bottom Line:
One such effector, SptP, functions early during pathogen entry to deactivate Rho GTPases and reverse pathogen-induced cytoskeletal changes following uptake.VCP and its adaptors p47 and Ufd1 were necessary for generating Salmonella-induced filaments on SCVs, a membrane fusion event characteristic of the pathogen replicative phase.Thus, Salmonella regulates the biogenesis of an intracellular niche through SptP-mediated dephosphorylation of VCP.

fig5: SptP-Dependent VCP Dephosphorylation during Infection(A) SptP interaction with VCP. HeLa cells were infected with S. Typhimurium ΔsptP expressing plasmid-encoded FLAG-tagged SptP variants (−, pSPTP, pR209A, pD441A). At 4 hr postinfection, cells were lysed with detergent, and SptP variants were immunoprecipitated from cell lysates with antibodies against SptP immobilized on protein G Sepharose. Eluted immunoprecipitates were assayed for presence of SptP and bound VCP and also supernatants for unbound VCP by immunoblotting with antibodies against FLAG (SptP) and VCP.(B) SptP-dependent dephosphorylation of VCP during S. Typhimurium cell infection. HeLa cells were individually infected with S. Typhimurium strains, described in (A), and wild-type (WT). At 4 hr postinfection, cells were lysed with detergent, and VCP was immunoprecipitated using antibodies against VCP immobilized on protein G Sepharose. Phosphorylation status of immunoprecipitated VCP was assessed by immunoblotting with antibodies against phosphotyrosine (VCPphos) and VCP serving as a loading control (VCP).(C) SptP-dependent syntaxin5 interaction with VCP during cell infection. HeLa cells transiently transfected with pcDNA3.1-HA-STX5 were either noninfected or infected in parallel with wild-type S. Typhimurium, the ΔsptP (−) mutant, or ΔsptP expressing plasmid-encoded SptP (pSPTP). HA-tagged syntaxin5 was immunoprecipitated from cell lysates with antibodies against HA immobilized on protein G Sepharose. Eluted immunoprecipitates were assayed for presence of syntaxin5 and bound VCP and also supernatants for unbound VCP by immunoblotting with antibodies against HA (syntaxin5) and VCP.

Mentions:
Next, we assessed the possibility that SptP delivered into mammalian cells specifically targets VCP. HeLa cells were infected in parallel with the ΔsptP mutant or with the same mutant expressing the FLAG-tagged SptP variants listed in Figure S2B. After 4 hr, cells were lysed with detergent and SptP isolated by immunoprecipitation (Figure 5A). Consistent with our earlier in vitro binding data (Figures 2A and 2B), VCP was coprecipitated (bound) by immobilized SptPD441A lacking PTPase activity, but not equivalently tagged SptPWT or GAP-defective SptPR209A, where VCP was only present in unbound fractions (Figure 5A). Parallel immunofluorescence revealed that, unlike SptPWT, the substrate-trap SptPD441A was absent from SCVs and localized diffusely throughout the perinuclear region (Figure S4A), implying that SptP may target VCP at membranes distinct from the SCV. Other persistent (FLAG-tagged) SPI-1 effectors, SopB and SipA (Brawn et al., 2007; Hernandez et al., 2004), did not bind VCP (Figure S4B).

fig5: SptP-Dependent VCP Dephosphorylation during Infection(A) SptP interaction with VCP. HeLa cells were infected with S. Typhimurium ΔsptP expressing plasmid-encoded FLAG-tagged SptP variants (−, pSPTP, pR209A, pD441A). At 4 hr postinfection, cells were lysed with detergent, and SptP variants were immunoprecipitated from cell lysates with antibodies against SptP immobilized on protein G Sepharose. Eluted immunoprecipitates were assayed for presence of SptP and bound VCP and also supernatants for unbound VCP by immunoblotting with antibodies against FLAG (SptP) and VCP.(B) SptP-dependent dephosphorylation of VCP during S. Typhimurium cell infection. HeLa cells were individually infected with S. Typhimurium strains, described in (A), and wild-type (WT). At 4 hr postinfection, cells were lysed with detergent, and VCP was immunoprecipitated using antibodies against VCP immobilized on protein G Sepharose. Phosphorylation status of immunoprecipitated VCP was assessed by immunoblotting with antibodies against phosphotyrosine (VCPphos) and VCP serving as a loading control (VCP).(C) SptP-dependent syntaxin5 interaction with VCP during cell infection. HeLa cells transiently transfected with pcDNA3.1-HA-STX5 were either noninfected or infected in parallel with wild-type S. Typhimurium, the ΔsptP (−) mutant, or ΔsptP expressing plasmid-encoded SptP (pSPTP). HA-tagged syntaxin5 was immunoprecipitated from cell lysates with antibodies against HA immobilized on protein G Sepharose. Eluted immunoprecipitates were assayed for presence of syntaxin5 and bound VCP and also supernatants for unbound VCP by immunoblotting with antibodies against HA (syntaxin5) and VCP.

Mentions:
Next, we assessed the possibility that SptP delivered into mammalian cells specifically targets VCP. HeLa cells were infected in parallel with the ΔsptP mutant or with the same mutant expressing the FLAG-tagged SptP variants listed in Figure S2B. After 4 hr, cells were lysed with detergent and SptP isolated by immunoprecipitation (Figure 5A). Consistent with our earlier in vitro binding data (Figures 2A and 2B), VCP was coprecipitated (bound) by immobilized SptPD441A lacking PTPase activity, but not equivalently tagged SptPWT or GAP-defective SptPR209A, where VCP was only present in unbound fractions (Figure 5A). Parallel immunofluorescence revealed that, unlike SptPWT, the substrate-trap SptPD441A was absent from SCVs and localized diffusely throughout the perinuclear region (Figure S4A), implying that SptP may target VCP at membranes distinct from the SCV. Other persistent (FLAG-tagged) SPI-1 effectors, SopB and SipA (Brawn et al., 2007; Hernandez et al., 2004), did not bind VCP (Figure S4B).

Bottom Line:
One such effector, SptP, functions early during pathogen entry to deactivate Rho GTPases and reverse pathogen-induced cytoskeletal changes following uptake.VCP and its adaptors p47 and Ufd1 were necessary for generating Salmonella-induced filaments on SCVs, a membrane fusion event characteristic of the pathogen replicative phase.Thus, Salmonella regulates the biogenesis of an intracellular niche through SptP-mediated dephosphorylation of VCP.